CN112159828B - Refractory branched glucan and processing method thereof - Google Patents
Refractory branched glucan and processing method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/60—Sweeteners
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12P19/08—Dextran
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention discloses refractory branched glucan and a processing method thereof, belonging to the technical field of health food processing. The invention comprises the following steps: weighing a certain mass of starch to prepare 10-40 wt% starch milk, and heating in a boiling water bath until complete gelatinization; cooling to 30-60 deg.C, adding 20-200U/gStarchThe alpha-amylase is subjected to heat preservation reaction for 0.1 to 3 hours, and then 20 to 200U/gStarchThe alpha-glucosidase is continuously reacted for 1 to 12 hours; inactivating enzyme, separating solid and liquid, and drying the obtained supernatant to obtain refractory branched dextran. The method disclosed by the invention is green and environment-friendly in process, simple in operation procedure and low in production cost, and the product has good probiotic physiological activity and can be used as novel dietary fiber to be applied to a plurality of fields such as health-care food, beverages and medicines.
Description
Technical Field
The invention relates to indigestible branched glucan and a processing method thereof, belonging to the technical field of health food processing.
Background
Starch is widely distributed in the plant world, is a main energy storage substance of seeds or fruits of grain crops, and is also a main raw and auxiliary material of modern food industry. As a typical component of daily rice and flour staple food for residents in China, starch is also the most main source of energy intake of organisms and accounts for 55-70% of the total energy intake. The natural cereal starch is generally considered as low-blood-sugar starch, but is easy to gelatinize and almost completely converted into digestible starch after the food is thermally processed, and belongs to high-blood-sugar food with quick energy release. The European and American transnational companies such as Ruian, Jiaji, Rogaiter and the like obtain novel products such as resistant starch, resistant dextrin and the like through chemical and physical modification and form large-scale production and sale; however, the research on starch nutritional products by domestic corn starch deep processing enterprises starts late, most of the corn starch deep processing enterprises are in the laboratory research stage, only a few companies start pilot-scale production, and the product performance has a larger gap compared with that of foreign countries. The existing commercial production method of branched glucan or resistant starch mainly comprises an acid-heating pyrodextrinization reaction and a starch sugar chain temperature-control crystallization method, but the methods have the bottleneck problems of complex reaction process, low preparation efficiency, poor stability, poor nutritional quality, high production cost and the like. Therefore, the problem that the quality of the existing product is difficult to meet the national big health industrial strategy and the individual requirements of consumers is solved, and the quality becomes the central importance of the field of the current starch nutritional products.
Disclosure of Invention
In order to solve the problems, the invention provides refractory branched glucan and a processing method thereof. The branch-shaped glucan which is difficult to digest is prepared by taking bulk starch as a substrate and adopting a multifunctional amylase coupling catalysis technology, and the prepared product has good probiotic physiological activity, including improving intestinal microecology, promoting nutrient absorption, enhancing immune system, preventing and relieving intestinal diseases and the like, and can be used as novel dietary fiber to be applied to the fields of health-care food, beverages, medicines and the like.
The first purpose of the invention is realized by the following technical scheme: a method of processing recalcitrant branched glucan, the method comprising the steps of: weighing a certain mass of starch to prepare 10-40 wt% starch milk, and heating in a boiling water bath until complete gelatinization; cooling to 30-60 deg.C, adding 20-200U/gStarchThe alpha-amylase is subjected to heat preservation reaction for 0.1 to 3 hours, and then 20 to 200U/gStarchThe alpha-glucosidase is continuously reacted for 1 to 12 hours; inactivating enzyme, separating solid and liquid, and drying the obtained supernatant to obtain refractory branched dextran.
In one embodiment of the invention, the alpha-amylase is a commercial amylase which functions to hydrolyze the starch chain and control the DE value between 3 and 10.
In one embodiment of the invention, the alpha-glucosidase is derived from an exoglycosidase of a microorganism of the GH13 family, said alpha-glucosidase is capable of transglycosidating to form a new alpha-1, 3 or alpha-1, 6 glycosidic bond.
In one embodiment of the present invention, the source of the α -glucosidase is a microorganism such as aspergillus niger, acremonium cladosporium, paecilomyces, or paenibacillus.
In one embodiment of the present invention, the starch is any one of corn starch, potato starch, tapioca starch, rice starch, wheat starch, etc.
In one embodiment of the present invention, the starch is any one of ordinary starch, waxy starch and high amylose starch.
In one embodiment of the invention, the inactivating enzyme is heat.
In one embodiment of the present invention, the solid-liquid separation is preferably centrifugal separation.
The second object of the present invention is to provide a hardly digestible branched glucan produced by the above-mentioned method for processing a hardly digestible branched glucan.
In one embodiment of the invention, the average molecular weight of the refractory branched dextran is 3200-8000 g/mol, wherein the content of alpha-1, 3 bonds is >7% and the content of alpha-1, 6 bonds is > 45%.
In one embodiment of the invention, the proportion of indigestible nutrient fragments in said indigestible branched glucan is > 75%.
It is a third object of the present invention to provide foods and drinks containing the above indigestible branched glucan.
The fourth object of the present invention is to provide the use of the indigestible branched glucan and the method for producing the indigestible branched glucan in the fields of health foods, beverages, medicines, and the like.
The invention has the following advantages:
1) the invention fully utilizes bulk starch rich in resources in China, designs an enzymatic processing system of refractory branched glucan, can greatly improve the nutritional quality of starch, and realizes green production of high-quality dietary fiber.
2) The method has simple steps and controllable reaction conditions, and realizes continuous and low-cost green production.
3) The invention obviously improves the bioavailability of the common starch, not only can be used as a functional raw material in the fields of food, medicine and the like, has wide potential market, but also can greatly improve the value of agricultural and sideline products, has important significance for improving the health level of people, and has obvious social benefit and economic benefit.
Drawings
FIG. 1 is a schematic diagram of the structure of the indigestible branched glucan prepared in example 2.
FIG. 2 NMR chart of indigestible branched dextran prepared in example 2.
Detailed Description
Molecular weight determination of branched dextran was determined by gel chromatography: an HPSEC-MALL-RI system is used for measuring the molecular weight of a sample, and Shodex OH-pak SB-806, 805 and 804 gel columns are adopted as chromatographic columns and are connected in series; NaNO3 solution with the mobile phase of 0.1 mol/L; the flow rate is 0.6 mL/min; the column temperature was 35 ℃.
Glycosidic bond types were determined by NMR methods: performing spectrogram analysis by using an AVANCE III-400 MHz nuclear magnetic resonance spectrometer, wherein the probe type is as follows: 5 mm PABBO-BB, pulse sequence: zg30, test temperature 70 ℃.
The proportion of indigestible nutrient fragments is determined by the Englyst method: weighing 150 mg of a sample, dissolving the sample in 10 mL of Na2HPO4-NaH2PO4 (100 mmol/L, pH 5.2) buffer solution, adding a proper amount of alpha-amylase and glucoamylase into the buffer solution, reacting the mixture at 37 ℃ and 150 r/min, taking out 0.1 mL of reaction solution every 20 min, mixing the reaction solution with 0.9 mL of absolute ethyl alcohol, inactivating enzyme, measuring the content of hydrolyzed glucose by using a glucose kit, and determining the content of indigestible nutrient fragments (= (total glucose of the sample-release amount of glucose in 120 min of hydrolysis) = 0.9.
Measurement of DE value after hydrolysis of starch chains by alpha-amylase: cu in the furin reagent can be reduced by reducing sugar2+Reduction to Cu+The end point of the reaction can be indicated by methylene blue, the content of reducing sugar is calculated according to the amount of reducing sugar required by a certain amount of the furin reagent for complete reduction, and the DE value is equal to that of a testRatio of sample reducing sugar to sample dry solids.
Alpha-amylase was purchased from novifin; corn starch, wheat starch, potato starch, corn waxy starch, rice high amylose starch were purchased from orycheng trade corn development ltd, respectively.
The present invention is further described below with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
The process for obtaining alpha-glucosidase is described in the literature, Microbial Starch-Converting Enzymes, percent instruments and Perspectives, Comprehensive Reviews in Food Science and Food Safety, 2018, 17 (5): 1238-1260, comprising the steps of enzyme gene excavation, engineering bacteria construction, enzyme production by fermentation and the like.
Specifically, the recombinant plasmid is obtained by connecting an alpha-glucosidase gene derived from a microorganism such as Aspergillus niger, Acremonium strictum, Paecilomyces variotii or Paenibacillus sp to an expression vector pET-15b (+). Transferring the recombinant plasmid into E.coil competent cells by adopting a chemical transformation method, adding 1 mL of LB culture medium, and culturing for 1-1.5 h in a shaking table at 37 ℃ and 200 r/min; the bacterial liquid of 100-. A single colony successfully transformed on an LB plate is selected and inoculated into an LB liquid culture medium containing 0.1 per mill (w/v) Amp, and is cultured for 12 hours in a shaking table at 37 ℃ and 200 r/min to serve as seed liquid. The seed solution is absorbed according to the inoculation amount of 2 percent and is inoculated in an LB culture medium of ammonia Amp to be expanded and cultured in a shaking table at 37 ℃ and 200 r/min. When the OD600 of the culture medium is between 0.6 and 0.8, IPTG is added into the culture medium until the final concentration is 0.1 mmol/L, the temperature is 16 ℃, and the induction expression is carried out for 20 hours at 200 r/min. And (3) centrifuging the induced bacterial liquid at 6000 r/min for 30 min, collecting thalli, performing ultrasonic treatment, centrifuging the crushed cell suspension (8000 r/min, 10 min and 4 ℃, and filtering the supernatant by using a 0.45-micron mixed cellulose ester microporous filter membrane, wherein the filtered enzyme liquid is crude enzyme liquid.
Example 2
Weighing 10g of corn starch to prepare starch milk with the mass percentage concentration of 10%, and placing the starch milk in a boiling water bath to be heated until the starch milk is completely gelatinized; when the temperature is reduced to 60 ℃, adding 200U/g of alpha-amylase of starch, carrying out heat preservation reaction for 0.5 h, and then adding 100U/g of alpha-glucosidase of starch, and continuing to react for 1 h; heating to inactivate enzyme, centrifuging, and drying the supernatant to obtain dendritic dextran product. In this process, the DE value after hydrolysis of the starch chain by alpha-amylase was 7.3.
Analysis and measurement show that the average molecular weight of the prepared branched glucan is 4800 g/mol, the content of alpha-1, 3 bonds is 8.2%, the content of alpha-1, 6 bonds is 49%, and the proportion of indigestible nutrient fragments reaches 80%. FIGS. 1 and 2 are a schematic diagram and an NMR chart of the hardly digested branched glucan prepared.
Example 3
Weighing 40g of wheat starch to prepare starch milk with the mass percentage concentration of 40%, and placing the starch milk in a boiling water bath to be heated until the starch milk is completely gelatinized; when the temperature is reduced to 30 ℃, adding 100U/g starch alpha-amylase, carrying out heat preservation reaction for 1h, and then adding 20U/g starch alpha-glucosidase, and continuing to react for 12 h; heating to inactivate enzyme, centrifuging, and drying the supernatant to obtain dendritic dextran product. In this process, the DE value after hydrolysis of the starch chain by alpha-amylase was 5.5.
Analysis and measurement show that the average molecular weight of the prepared branched glucan is 6100 g/mol, the content of alpha-1, 3 bonds is 7.5%, the content of alpha-1, 6 bonds is 60%, and the proportion of indigestible nutrient fragments is 83%.
Example 4
Weighing 20g of potato starch to prepare 20% starch milk in mass percentage concentration, and heating in a boiling water bath until complete gelatinization; when the temperature is reduced to 50 ℃, adding 20U/g of alpha-amylase of starch, carrying out heat preservation reaction for 3 hours, and then adding 200U/g of alpha-glucosidase of starch, and continuing to react for 1 hour; heating to inactivate enzyme, centrifuging, and drying the supernatant to obtain dendritic dextran product. In this process, the DE value after hydrolysis of the starch chain by alpha-amylase was 3.7.
The average molecular weight of the branched glucan obtained by the analysis and measurement was 4200 g/mol, the content of α -1,3 bonds was 7.7%, the content of α -1,6 bonds was 53%, and the content of indigestible nutrient fragments was 78%.
Example 5
Weighing 10g of corn waxy starch to prepare starch milk with the mass percentage concentration of 10%, and placing the starch milk in a boiling water bath to be heated until the starch milk is completely gelatinized; when the temperature is reduced to 60 ℃, adding 200U/g starch alpha-amylase, keeping the temperature and reacting for 0.5 h, and then adding 100U/g starch alpha-glucosidase, and continuing to react for 1 h; heating to inactivate enzyme, centrifuging, and drying the supernatant to obtain dendritic dextran product. In this process, the DE value after hydrolysis of the starch chain by alpha-amylase was 4.9.
Analysis and measurement show that the average molecular weight of the prepared branched glucan is 5400 g/mol, the content of alpha-1, 3 bonds is 7.6 percent, the content of alpha-1, 6 bonds is 53 percent, and the proportion of indigestible nutrient fragments reaches 78 percent.
Example 6
Weighing 10g of corn high amylose starch to prepare starch milk with the mass percentage concentration of 10%, and heating in a boiling water bath until complete gelatinization; when the temperature is reduced to 60 ℃, adding 200U/g starch alpha-amylase, keeping the temperature and reacting for 0.5 h, and then adding 100U/g starch alpha-glucosidase, and continuing to react for 1 h; heating to inactivate enzyme, centrifuging, and drying the supernatant to obtain dendritic dextran product. In this process, the DE value after hydrolysis of the starch chain by alpha-amylase was 3.9.
Analysis and measurement show that the average molecular weight of the prepared branched glucan is 3700 g/mol, the content of alpha-1, 3 bonds is 7.9%, the content of alpha-1, 6 bonds is 46%, and the proportion of indigestible nutrient fragments is 85%.
Comparative example 1
Weighing 10g of corn starch to prepare 10 mass percent starch milk, and heating the starch milk in a boiling water bath until the starch milk is completely gelatinized; when the temperature is reduced to 60 ℃, adding 200U/g starch alpha-amylase and reacting for 0.5 h under heat preservation; heating to inactivate enzyme, centrifuging, and drying the supernatant to obtain dendritic dextran product.
Analysis and measurement show that the average molecular weight of the prepared branched glucan is 4840 g/mol, the content of alpha-1, 3 bonds is 0%, the content of alpha-1, 6 bonds is 9%, and the proportion of indigestible nutrient fragments reaches 12%.
Comparative example 2
Weighing 10g of corn starch to prepare starch milk with the mass percentage concentration of 10%, and placing the starch milk in a boiling water bath to be heated until the starch milk is completely gelatinized; when the temperature is reduced to 60 ℃, adding alpha-glucosidase of 100U/g starch for reaction for 1 h; heating to inactivate enzyme, centrifuging, and drying the supernatant to obtain dendritic dextran product.
Analysis and measurement show that the average molecular weight of the prepared branched glucan is 4930 g/mol, the content of alpha-1, 3 bonds is 0%, the content of alpha-1, 6 bonds is 9%, and the proportion of indigestible nutrient fragments reaches 11%.
Comparative example 3
Weighing 10g of corn starch to prepare starch milk with the mass percentage concentration of 10%, and placing the starch milk in a boiling water bath to be heated until the starch milk is completely gelatinized; when the temperature is reduced to 60 ℃, simultaneously adding 200U/g starch alpha-amylase and 100U/g starch alpha-glucosidase for reacting for 1 h; heating to inactivate enzyme, centrifuging, and drying the supernatant to obtain dendritic dextran product.
Analysis and measurement show that the average molecular weight of the prepared branched glucan is 5000 g/mol, the content of alpha-1, 3 bonds is 3 percent, the content of alpha-1, 6 bonds is 24 percent, and the proportion of indigestible nutrient fragments is 39 percent.
Comparative example 4
Weighing 10g of corn starch to prepare starch milk with the mass percentage concentration of 10%, and placing the starch milk in a boiling water bath to be heated until the starch milk is completely gelatinized; when the temperature is reduced to 60 ℃, adding 200U/g starch alpha-amylase, keeping the temperature and reacting for 0.05 h, and then adding 100U/g starch alpha-glucosidase, and continuing to react for 1 h; heating to inactivate enzyme, centrifuging, and drying the supernatant to obtain dendritic dextran product. In this process, the DE value after hydrolysis of the starch chain by alpha-amylase was 2.1.
Analysis and measurement show that the average molecular weight of the prepared branched glucan is 4800 g/mol, the content of alpha-1, 3 bonds is 5%, the content of alpha-1, 6 bonds is 32%, and the proportion of indigestible nutrient fragments reaches 43%.
Comparative example 5
Weighing 10g of corn starch to prepare starch milk with the mass percentage concentration of 10%, and placing the starch milk in a boiling water bath to be heated until the starch milk is completely gelatinized; when the temperature is reduced to 60 ℃, adding 200U/g starch alpha-amylase, keeping the temperature and reacting for 5 hours, and then adding 100U/g starch alpha-glucosidase, and continuing to react for 1 hour; heating to inactivate enzyme, centrifuging, and drying the supernatant to obtain dendritic dextran product. In this process, the DE value after hydrolysis of the starch chain by alpha-amylase was 12.5.
Analysis and measurement show that the average molecular weight of the prepared branched glucan is 3200 g/mol, the content of alpha-1, 3 bonds is 4 percent, the content of alpha-1, 6 bonds is 37 percent, and the proportion of indigestible nutrient fragments reaches 50 percent.
Comparative example 6
Referring to example 1, the amount of alpha-amylase used was 200U/g and the amount of alpha-glucosidase used was 100U/g, respectively, which were 10U/g and 500U/g, respectively, to obtain corresponding branched glucan products. The performance results of the scored dendritic dextran product are shown in table 1.
TABLE 1 results for indigestible nutritional fraction obtained with different amounts of multifunctional enzyme system
| Dosage (U/g) | Indigestible nutritional fraction |
| 10U/g of alpha-amylase and 100U/g of alpha-glucosidase | 17% |
| 200U/g of alpha-amylase and 500U/g of alpha-glucosidase | 25% |
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A method of processing refractory branched glucan, comprising the steps of: weighing a certain mass of starch to prepare 10-40 wt% starch milk, and heating in a boiling water bath until complete gelatinization; cooling to 30-60 deg.C, adding 20-200U/gStarchThe alpha-amylase is subjected to heat preservation reaction for 0.1 to 3 hours, and then 100-200U/gStarchThe alpha-glucosidase is continuously reacted for 1 hour, or 20U/gStarchThe alpha-glucosidase continuously reacts for 12 hours; deactivating enzyme, performing solid-liquid separation, and drying the obtained supernatant to obtain refractory branched glucan; the alpha-glucosidase is derived from exoglycosidase of a GH13 family microorganism, is derived from Aspergillus niger, Acremonium strictum, Paecilomyces variotii or Paenibacillus, and can be used for glucoside conversion to form new alpha-1, 3 and alpha-1, 6 glycosidic bonds; controlling the DE value of alpha-amylase after hydrolyzing the starch chain to be 3-10.
2. The method as claimed in claim 1, wherein the starch is selected from the group consisting of corn starch, potato starch, tapioca starch, rice starch, and wheat starch.
3. The method as claimed in claim 2, wherein the starch is any one of normal starch, waxy starch and high amylose starch.
4. The indigestible branched glucan produced by the method for processing indigestible branched glucan according to any one of claims 1 to 3.
5. The indigestible branched glucan according to claim 4, wherein the average molecular weight of the indigestible branched glucan is 3200-8000 g/mol, wherein the α -1,3 bond content is >7% and the α -1,6 bond content is > 45%.
6. Indigestible branched glucan according to claim 4 or 5, wherein the proportion of indigestible nutrient fragments in the indigestible branched glucan is > 75%.
7. Food or drink comprising the indigestible branched glucan according to any one of claims 4 to 6.
8. The method for processing indigestible branched glucan according to any one of claims 1 to 3 or the use of the indigestible branched glucan according to any one of claims 4 to 6 in the field of health foods and beverages.
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